Mitochondrial oxidative stress is associated with ischemia-reperfusion (I/R) injury. At the myocardial level of the post-ischemic heart, defective energy metabolism has been linked to marked overproduction of oxygen free radical(s). Available evidence indicates that [a] specific interactions between oxygen free radicals and respiratory chain proteins are critical in the I/R pathophysiology, and [b] oxygen free radical- induced oxidative stress can act as a redox signal to change the thiol redox state of mitochondria! proteins, subsequently triggering cellular events such as apoptosis, proliferation and senescence. Two mitochondrial proteins containing critical redox thiols, NADH dehydrogenase (NDH) and succinate ubiquinone reductase (SQR), are also catalytic components responsible for superoxide production in mitochondria. The broad long-term goal of this project is to elucidate the molecular mechanism of how oxygen free radical production in mitochondria contributes to the myocardial injury. The key hypotheses of superoxide generation and protein S-glutathiolation will be tested by pursuing the following specific aims using the isolated enzyme complex and post-ischemic hearts. [1] Determine the mechanism of superoxide production by NDH. [2] Determine the mechanism of superoxide production by SQR. [3] Elucidate the molecular mechanism of protein S-glutathiolation resulting from the major pathway of oxygen free radical production. [4] Evaluate protein S-glutathiolation and NDH/SQR-derived S- glutathiolation in post-ischemic hearts. Enzyme-mediated superoxide production will be measured by EPR spin trapping and immobilized enzyme EPR (IE-EPR). Enzyme-mediated NO consumption rates will be measured by electrochemical detection using a NO electrode. Protein-derived S-glutathiolation and protein radical formation will be detected by immunoblotting. Protein radical(s), reactive thiol(s), and glutathione-binding (GS-binding) domain(s) will be identified by mass spectrometry and peptide mapping. The functional role of each identified domain will be defined by immunochemistry using specific antibodies against conformational epitope peptides. The utilization of protein S-glutathiolation as a biomarker of redox change resulting from oxidative stress will be evaluated in the systems of post- ischemic heart. Results from this proposal will provide fundamental information concerning the pathophysiological role of oxygen free radicals and protein redox thiol(s) in mitochondrial dysfunction, and potentially improve clinical diagnosis, therapeutics, and prevention of heart diseases.